Crystalline 1-kestose and process for preparing the same

ABSTRACT

A process for producing crystal 1-kestose wherein granular crystal 1-kestose in the form of large crystals can be produced at a high yield is disclosed. A highly pure solution of 1-kestose is concentrated to a Brix of 75 or higher; either seed crystals are added, or the solution is vacuum-concentrated to generate microcrystals for use as nuclei; then, a crystal growing step by vacuum-concentration and a microcrystal dissolving step for redissolving microcrystals which have formed in the concentrate are repeated at least twice each. Alternatively, a highly pure solution of 1-kestose is concentrated to a Brix of 80 or higher; either seed crystals are added, or the solution is allowed to initiate crystallization; after crystals are allowed to grow, a cooling step where the temperature is lowered by 5° C. to 20° C. from the previous step and a crystal growing step where the concentrate is maintained at the temperature to allow the crystals to grow are repeated at least twice each. Also, an enzyme for producing 1-kestose efficiently from sucrose is disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for producing crystal1-kestose, specifically a process for producing large crystal grains ofcrystal 1-kestose by vacuum crystallization or cold crystallization at ahigh yield. The present invention also relates to an enzyme forefficiently producing 1-kestose, and a process for producing crystal1-kestose using said enzyme.

2. Description of the Related Art

The molecular structure of 1-kestose, a type of trisaccharide, is thesame as that of sucrose except that the fructose half of 1-kestose iscoupled with another fructose at position 1 via a β-2,1 bond.Fructooligosaccharides are characterized by little cariosity,indigestibility to biological digestive enzymes, and the specificfacilitation of the growth of Bifidobacterium in the intestines, asdemonstrated by some of the present inventors of the present invention(Japanese Patent Publication No. 53834/1984). It is believed that1-kestose, which is a component of fructooligosaccharides, also has thesame characteristics.

Commercially available crystalline oligosaccharides have been limited toraffinose, which has small crystal grain sizes of about 0.1 to 0.3 mmlengthwise. The currently most well-known crystalline sugar, sucrose, isproduced mostly as granulated sugar, which is composed primarily oflarge crystals of about 0.25 to 1 mm, and soft sugar, which is a mixtureof large and medium crystals of about 0.1 to 0.5 mm. Inoligosaccharides, large grains are also favored for high utility.

Although both 1-kestose and sucrose have columnar crystals, the crystalsof 1-kestose are more rectangular than the almost cubic crystals ofsucrose. Therefore, 1-kestose should have larger crystals than sucrosein order to show physical properties similar to those of granulatedsugar.

In a previously disclosed method for producing crystal 1-kestose,aqueous solution of 1-kestose of Brix 85 or higher and a purity of 70%or higher is heated to a temperature of 80° C. or higher; a suspensionof microcrystals is added to the solution; the temperature is lowered to65 to 75° C. to deposit crystals; and, while the temperature ismaintained at 60 to 80° C., the deposited crystal 1-kestose is recovered(Japanese Patent Publication No. 70075/1994).

This method has a disadvantage in that the yield of crystals is low atabout 30 to 40% because the concentration and purity of thecrystallizing solution drop as crystals deposit. Further, this methodunavoidably involves the deposition of microcrystals with increasing theviscosity of the solution. Thus, the solution should be heated duringthe recovery of crystals. Further, the grain sizes of the obtainedcrystals are not uniform and generally small.

On the other hand, 1-kestose may be produced from sucrose, making use ofthe activity of fructose transferase. In one of such known methods,sucrose solution adjusted to a concentration of 50% or higher is allowedto react at pH 4.0 to 7.0 in the presence of an enzyme having fructosetransferase activity which is derived from an Asperaillus or Fusariummicroorganism (Japanese Patent Publication No. 62184/1988). In anotherknown method, an enzyme having fructose transferase activity derivedfrom an AsTeraillus microorganism is allowed to react at pH 4.0 to 7.0,a temperature of 25 to 65° C., and a sucrose concentration of 20 to 70%(Japanese Patent Laid-Open Publication No. 268190/1986).

However, these methods are aimed primarily to obtain a mixture of atrisaccharide (1-kestose), a tetrasaccharide (nystose), and apentasaccharide (fructosyl nystose), i.e., fructooligosaccharides, fromsucrose. The rates of conversion from sucrose to 1-kestose in thismethod are only 36 to 41% at maximum (refer to Table 3 in JapanesePatent Publication No. 62184/1988, and Examples in Japanese PatentLaid-Open Publication No. 268190/1986). Also, in these methods, 11 to23% of nystose is produced along with 1-kestose, leaving 11 to 23% ofnon-reacted sucrose, while 13 to 32% of monosaccharides such as fructoseor glucose are produced as byproducts (ibid.).

It should be noted that the presence of monosaccharides, sucrose, andnystose is disadvantageous for the following reason: In thetwo-component simulated-moving bed chromatographic separation, which isused generally for producing isomerized sugar, etc., a solutioncontaining two or more sugars of different molecular weights is dividedinto two fractions according to the difference in their molecularweights. If this method is used to obtain 1-kestose of high purity froma solution containing 1-kestose, the solution should be either (1)divided into two fractions, i.e., a fraction containing sugars withlower molecular weights than 1-kestose, and one containing those withmolecular weights equal to or higher than that of 1-kestose, including1-kestose; or (2) divided into two fractions, i.e., a fractioncontaining sugars with higher molecular weights than 1-kestose, and onecontaining those with molecular weights equal to or lower than that of1-kestose, including 1-kestose. In the former case, for example, as thefraction contains sugars of higher molecular weights than that of1-kestose, the purity of 1-kestose in the fraction is inevitably lowdepending on the content of such sugars. When the solution contains highcontents of sugars whose molecular weights are higher than that of1-kestose, e.g., nystose, with the two-component simulated-moving bedchromatographic separation, two or more steps of operations arerequired, such as a second chromatographic separation of the resultantfraction, in order to obtain 1-kestose solution at a high purity (e.g.,80% or higher), which is necessary for crystallization. In the lattercase, the process has the same disadvantage when the contents ofmonosaccharides and sucrose are high. Therefore, a need exists for amethod using enzymatic reactions in which the production ofmonosaccharides, sucrose, nystose, etc., is reduced, that is, a methodin which 1-kestose can be selectively produced, and the conditions and anovel enzyme for this method.

Among Penicillium microorganisms, it has been suggested that Penicilliumfrequentans has an enzyme having fructose transferase activity (JapanesePatent Laid-Open Publication No. 293494/1992). However, this report onlystates that the microorganism produces fructose transferase, does notmention the rate of conversion to 1-kestose.

A method for selectively producing 1-kestose using a Scopulariopsismicroorganism that produces 1-kestose in the presence of sucrose whileconsuming glucose has been reported (Japanese Patent Publication No.47197/1993, Japanese Patent Publication No. 41600/1992). After themicroorganism is incubated in a medium containing sucrose to produce1-kestose, 1-kestose is recovered from the culture. According to thepublication, the rate of conversion from sucrose to 1-kestose is as highas 60%. However, since the method uses the whole fungus body, the totalsugar concentration in the culture, i.e., the sucrose concentration atthe beginning of incubation, should be as low as about 15%. In addition,high contents of proteins and other impurities should be removed duringpurification.

Thus, methods for producing 1-kestose more selectively, particularly anovel enzyme and novel conditions for reaction, are solicited.

SUMMARY OF THE INVENTION

Inventors have now found that the viscosity of the fraction can be madelow enough to enable the separation of crystals as solids from thesolution at room temperature without heating the solution, by acontrolled concentration procedure and temperature control based on whatis called vacuum crystallization or cold crystallization. It has also befound that larger crystals of 1-kestose are obtained at a high yield.The inventors have also found certain microorganisms produce enzymeswhich efficiently produce 1-kestose. The present invention is based onthese findings.

Thus, the object of the present invention is to provide a process forproducing crystal 1-kestose wherein crystal 1-kestose is obtained at ahigh yield by the separation of solid from liquid at room temperature.Another object of the present invention is to provide a process forproducing crystal 1-kestose wherein large crystals of crystal 1-kestose,preferably in a granular form, are obtained.

Still another object of the present invention is to provide largecrystals of 1-kestose in a granular form.

According to the first aspect of the present invention, there isprovided a process for producing crystal 1-kestose comprising the stepsof:

(a) concentrating a highly pure 1-kestose solution having 80% or more of1-kestose to a Brix of 75 or higher, adding seed crystals, and thenheating the resultant concentrate to 60° C. or higher to allow crystalsto grow,

(b) as a crystal growing step, concentrating the concentrate under areduced pressure to allow crystals to grow, and

(c) as a microcrystal dissolving step following the crystal growingstep, heating the concentrate to redissolve microcrystals which havebeen generated in the concentrate and, if the microcrystals fail toredissolve completely, adding water to the concentrate to dissolve themicrocrystals,

followed by repeating at least once each of the (b) crystal growing stepand the (c) microcrystal dissolving step, and

recovering crystal 1-kestose.

In addition there is provided a modified process of that according tothe first aspect of the present invention comprising the steps of:

instead of steps (a) and (b) above,

(a′) concentrating a highly pure 1-kestose solution having 80% or moreof 1-kestose to a Brix of 75 or higher, and heating the resultantconcentrate to 60° C. or higher, and

(b′) concentrating the concentrate under a reduced pressure to lower thetemperature of the concentrate and to initiate crystallization and allowthe resultant crystals to grow,

followed by the (c) microcrystal dissolving step as defined above,

then by repeating at least once each of the (b) crystal growing step andthe (c) microcrystal dissolving step as defined above, and

recovering crystal 1-kestose.

According to the second aspect of the present invention, there isprovided a process for producing crystal 1-kestose comprising the stepsof:

(α) concentrating a highly pure 1-kestose solution having 80% or more of1-kestose to a Brix of 80 or higher, heating the solution to 70 to 95°C., adding seed crystals, and maintaining the resultant concentrate inthe temperature range to allow the crystals to grow,

(β) as a succeeding cooling step, cooling the concentrate by 5 to 20° C.from the temperature in the previous step, and

(γ) as a crystal growing step, maintaining the concentrate at thelowered temperature of the cooling step above to allow the crystals togrow,

followed by repeating at least once each of the (β) cooling step and the(γ) crystal growing step,

lowering the temperature to 20 to 60° C., and recovering crystal1-kestose.

In addition, there is provided a modified process of that according tothe second aspect of the present invention comprising the steps of:

instead of step (α) above,

(α′) concentrating a highly pure 1-kestose solution having 80% or moreof 1-kestose to a Brix of 80 or higher, maintaining the temperature ofthe solution in the range of 50 to 60° C. to initiate crystallization,and then maintaining the resultant crystals at 70 to 95° C. to grow,

followed by repeating at least once each of the (β) cooling step and the(γ) crystal growing step,

lowering the temperature to 20 to 60° C., and recovering crystal1-kestose.

According to the present invention, there is also provided a crystal1-kestose in the form of columnar crystal having a length of 0.3 mm orlonger (preferably 0.3 to 2 mm) and a purity of 95% or higher.

Further, according to the third aspect of the present invention, thereis provided a process for producing crystal 1-kestose comprising thesteps of:

(i) either of

(1) allowing an enzyme having fructose transferase activity derived froman Aspergillus microorganism to react on a sucrose solution to produce1-kestose accounting for 43 wt % or more of the resultant sugars,

(2) allowing an enzyme having fructose transferase activity derived froma Penicillium microorganism to react on a sucrose solution to produce1-kestose accounting for 46 wt % or more of the resultant sugars, or

(3) allowing an enzyme having fructose transferase activity derived froma Scopulariopsis microorganism to react on a sucrose solution to produce1-kestose accounting for 53 wt % or more of the resultant sugars;

(ii) isolating from 1-kestose obtained in step (i) a fraction having apurity of 80% or higher by chromatographic separation; and

(iii) crystallizing 1-kestose fraction obtained in step (ii) to producecrystal 1-kestose in the form of columnar crystal having a length of 0.3mm or longer and a purity of 95% or higher.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Process of the First and SecondAspects of the Present Invention

Crystallizing Sample

Both in the first and second aspects of the present invention, acrystallizing sample containing a highly pure 1-kestose is used as astarting material. A higher 1-kestose purity would be more advantageous,preferably 80% or higher in the process according to the presentinvention, and more preferably 90% or higher. The purity of 1-kestoseherein refers to the content of 1-kestose expressed as the weightpercentage of the sugar content in the crystallizing sample.

To prepare a crystallizing sample of 1-kestose, a sugar sourcecontaining 1-kestose is either prepared or obtained, and purified toincrease its purity of 1-kestose. A sugar source containing 1-kestosemay be prepared, for example, by allowing fructose transferase derivedfrom a plant, a microorganism, etc., to react on sucrose or othersubstrate; by incubating a microorganism in a medium containing sucroseor other sugar; or from various commercially available mixtures offructooligosaccharides by standard techniques such as chromatographicseparation or membrane separation, or Steffen process, in which lime isused to precipitate impurities.

According to the preferred embodiment of the present invention, theproportion of nystose in the sugar content except 1-kestose in thecrystallizing sample of 1-kestose should be minimal. Inventors havefound that the nystose content in the crystallizing sample should belower to obtain larger 1-kestose crystals. More specifically, it hasbeen demonstrated that nystose, if exists, inhibits the crystallizationof 1-kestose. The preferable nystose content is 10 wt % or less of1-kestose.

This finding applies not only to the processes according to the firstand second modes the present invention, but to previously knownprocesses for producing 1-kestose as well.

Process of the First Aspect of the Present Invention

In the process according to the first aspect of the present invention,the crystallizing sample is first concentrated to a Brix of 75 orhigher, preferably 80 to 85. Concentration may be performed by heatingor decompression. Then, seed crystals are added to the concentrate. Thepreferable quantity of the seed crystals is 1 wt % or less of the solidscontent in the concentrate. After the seed crystals are added, theconcentrate is allowed to continue to crystallize with the seed crystalsas nuclei. It is preferable that the temperature of the concentrate bemaintained at 60° C. or higher for about 0.5 to 6 hours, more preferablyat 70 to 95° C. for 1 to 3 hours.

In another preferred embodiment of the present invention, crystal nucleimay be formed without adding seed crystals unlike the above procedure.The concentrate may be concentrated under a reduced pressure at atemperature of 60° C. or higher to deposit microcrystals, then allowedto continue to crystallize under the conditions above described with themicrocrystals as nuclei. It is preferable that microcrystals be formedin the range of 10 to 160 mmHg in absolute pressure and 30 to 70° C. intemperature, more preferably in the range of 40 to 120 mmHg in absolutepressure and 50 to 60° C. in temperature.

After the concentrate is allowed to continue to crystallize as describeabove, it is concentrated under a reduced pressure, preferably at anabsolute pressure of 40 to 200 mmHg for 10 to 60 minutes, morepreferably at an absolute pressure of 60 to 120 mmHg for 30 to 60minutes. Through the concentration procedure, the temperature of theconcentrate drops to 40 to 70° C., facilitating the growth of crystals(crystal growing step).

Following the crystal growing step, the concentrate is subjected to themicrocrystal dissolving step described below. While the growth of1-kestose crystals is observed as a result of vacuum concentration, theconcentration procedure generally produces 1-kestose microcrystals aswell. The microcrystals are redissolved by heating the concentratepreferably at 70° C. to 95° C., more preferably at 75 to 85° C., andmaintaining the temperature for 0.5 to 6 hours, preferably 1 to 2 hours.If heating fails to redissolve the microcrystals completely, water isadded to redissolve preferably the entire microcrystals.

In the process according to the first aspect, the microcrystaldissolving step is followed by the repetition of at least once each ofthe same crystal growing step and microcrystal dissolving step asdescribed above. In particular, after the concentrate is maintainedpreferably at an absolute pressure of 40 to 200 mmHg for 10 to 60minutes to allow the crystals to grow, the resultant microcrystals areredissolved by heating the concentrate preferably at 70° C. to 95° C.and maintaining the temperature for 0.5 to 6 hours. If the heating failsto redissolve the microcrystals completely, water is added to redissolvepreferably the entire microcrystals.

The crystals of 1-kestose are allowed to grow sufficiently to a desiredgrain size in these steps. The obtained crystals may be isolated andrecovered by standard techniques (e.g., by centrifugation). Thepreferred embodiment of the present invention has a significantadvantage that, after crystallization is over, the concentrate, in whichthe crystals of 1-kestose have been allowed to grow sufficiently to agranular form, has a viscosity low enough to enable the recovery of thecrystals without heating the solution. After recovery, the crystals maybe dried as necessary to finally make crystal 1-kestose.

According to the first aspect of the present invention, 1-kestose isobtained at a yield as high as 50 to 60%.

According to the preferred embodiment of the present invention, crystal1-kestose in the form of columnar crystals having lengths of 0.3 mm orlonger (preferably 0.3 to 2 mm, more preferably 0.5 to 1.2 mm) and apurity of 95% or higher can be obtained. Specifically, by controllingthe Brix to about 75 to 85, preferably about 79 to 83, in the crystalgrowing step described above, generation of microcrystals in theconcentrate is inhibited, thereby facilitating the growth of crystals,to efficiently produce the large grains of crystal 1-kestose asdescribed above.

In addition, in the method of the first aspect of the present invention,the crystallizing sample may be replenished to make up for the loss involume caused in the crystal growing step above, after the crystalgrowing step and before the microcrystal dissolving step, or after themicrocrystal dissolving step and before the second crystal growing step.This replenishment would help increase the volume processed peroperation and, at the same time, maintain the high purity of 1-kestosein the solution, which may otherwise drop as crystallization proceedthereby, enabling crystallization to take place at a consistently high1-kestose purity.

Crystal 1-kestose may be prepared in continuous processes by repeatingthe same procedure as described above or carrying out the procedureaccording to the second aspect as described below on the sugar solutioncontaining 1-kestose from which crystal 1-kestose has been recovered.Alternatively, the same procedure as described above or the procedureaccording to the second aspect as described below may be repeated on thesugar solution containing 1-kestose from which crystal 1-kestose hasbeen recovered after, as necessary, replenishing the solution with freshcrystallizing sample. These continuous procedures of crystallizationwould help further increase the yield of 1-kestose crystal, in somecases, to a total yield as high as 80% or higher.

Process of the Second Aspect of the Present Invention

In the process according to the second aspect of the present invention,the crystallizing sample is first concentrated to a Brix of 80 orhigher, preferably 83 to 88. Concentration may be performed by heatingor decompression. Then, while the concentrate is maintained at atemperature of 70 to 95° C., seed crystals are added to the concentrate.The preferable quantity of the seed crystals is 1 wt % or less of thesolids content in the concentrate.

In another preferred embodiment of the present invention, crystal nucleican be formed, without adding seed crystals unlike the above procedure.Interestingly, inventors have found that microcrystals, which functionas crystal nuclei, can be formed efficiently by heating the concentrate,which has been concentrated to a Brix of 80 or higher, from around roomtemperature (about 25° C.) to 50 to 60° C., and that the number and sizeof the microcrystals can be controlled by regulating the rate ofheating. Higher rate of heating from room temperature to the temperaturerange specified above would result in a smaller number of microcrystalsas crystal nuclei, thus resulting in large crystals being formed.Furthermore, lower rate of heating would result in a greater number ofmicrocrystals as crystal nuclei, thus resulting in small crystals.

After seed crystals are added or microcrystals which function as crystalnuclei are formed as described above, the concentrate is maintained hotat 70 to 95° C., preferably at 75 to 85° C., for 30 minutes to 6 hoursto allow the crystals to grow.

In the next cooling step of this process, the concentrate is cooled tolower its temperature by 5 to 20° C., preferably by about 10° C., fromthe previous step. Then, the concentrate is maintained hot at thetemperature for 30 minutes to 6 hours to allow the crystals to grow in asubsequent crystal growing step.

In this process, the cooling step and the crystal growing step arefollowed by the repetition of at least once each of the same coolingstep and crystal growing step. In other words, the temperature of theconcentrate is lowered from the previous step by 5 to 20° C., preferablyby about 10° C., then maintained high at the same temperature for 30minutes to 6 hours to allow the crystals to grow.

If a large amount of microcrystals have formed during crystallization,it is recommended that the solution temperature be increased preferablyby about 10 to 20° C. from that when microcrystals formed, in order toredissolve the microcrystals. Then the cooling step and the crystalgrowing step are carried out again.

The cooling step and the crystal growing step are repeated until thetemperature of the crystallizing solution is 20 to 60° C., preferablyabout 40 to 50° C.

The crystals of 1-kestose are allowed to grow sufficiently to a desiredgrain size in these steps. The obtained crystals may be isolated andrecovered by standard techniques (e.g., by centrifugation). Thepreferred embodiment of the present invention has a significantadvantage as does the process of the first aspect of the presentinvention. After crystallization is over, the concentrate containing thecrystals of 1-kestose which have been allowed to grow sufficiently to agranular form, has a viscosity low enough to enable the recovery ofcrystals without heating the solution. After recovery, the crystals maybe dried as necessary to finally make crystal 1-kestose.

According to the second aspect of the present invention, 1-kestose isobtained at a yield of 40% or higher, or as high as 43 to 55% in apreferred mode.

According to the preferred embodiment, as the process of the firstaspect of the present invention, crystal 1-kestose in the form ofcolumnar crystals having lengths of 0.3 mm or longer (preferably 0.3 to2 mm, more preferably 0.5 to 1.2 mm) and a purity of 95% or higher.

Furthermore, as in the process of the first aspect of the presentinvention, crystal 1-kestose may be prepared in continuous processes byrepeating the same procedure as described above or carrying out theprocedure of the first aspect of the present invention on the sugarsolution containing 1-kestose from which crystal 1-kestose has beenrecovered. Alternatively, the same procedure as described above or theprocedure of the first aspect of the present invention may be repeatedon the sugar solution containing 1-kestose from which crystal 1-kestosehas been recovered after replenishing the solution with freshcrystallizing sample. These continuous procedures of crystallizationwould help further increase the yield of 1-kestose crystals.

Process of the Third Aspect of the Present Invention

According to the third aspect of the present invention, there isprovided a process for producing 1-kestose from sucrose using an enzymederived from a certain microorganism.

Regarding the third aspect of the present invention, 1 unit of enzymehaving fructose transferase activity refers to the following meaning:

In the case of an enzyme derived from an Asperaillus microorganism, thequantity of enzyme which can produce 1 μmol of 1-kestose in 1 minute at40° C., pH 5.0, and a substrate (sucrose) concentration of 10 wt %;

In the case of an enzyme derived from a Penicillium microorganism, thequantity of enzyme which can produce 1 μmol of 1-kestose in 1 minute at50° C., pH 7.0, and a substrate (sucrose) concentration of 10 wt %; and

In the case of an enzyme derived from a Scopulariopsis microorganism,the quantity of enzyme which can produce 1 μmol of 1-kestose in 1 minuteat 40° C., pH 7.0, and a substrate (sucrose) concentration of 10 wt %.

Step (i): Synthesis of 1-Kestose from Sucrose

In the process according to the third aspect of the present invention,sucrose is first converted to 1-kestose in either of the followingsteps:

(i) (1) allowing an enzyme having fructose transferase activity derivedfrom an Aspergillus microorganism to react on a sucrose solution toproduce 1-kestose accounting for 43 wt % or more of the resultantsugars;

(2) allowing an enzyme having fructose transferase activity derived froma Penicillium microorganism to react on a sucrose solution to produce1-kestose accounting for 46 wt % or more of the resultant sugars; or

(3) allowing an enzyme having fructose transferase activity derived froma Scopulariopsis microorganism to react on a sucrose solution to produce1-kestose accounting for 53 wt % or more of the resultant sugars.

Step (i)(1)

In step (i)(1), an enzyme having fructose transferase activity which canbe prepared from an Asperaillus microorganism is used. The enzyme can beprepared preferably from Aspergillus niger, specifically fromAspergillus niger ATCC20611.

The enzyme can be recovered from a culture by incubating themicroorganism in a suitable medium (e.g., a medium containing 3.0 to10.0% of sucrose, 3.0 to 10.0% of yeast extract, and 0.3 to 1.0% of CMC)at initial pH of 6.0 to 7.0 and a temperature of 25 to 30° C. for 48 to96 hours.

The enzyme acts on a sucrose solution with a concentration of 50 wt % orhigher, preferably at a rate of 0.5 to 100 units per 1 g of sucrose, atpH 5.0 to 10.0 and a temperature of 50 to 65° C. and produces 1-kestoseaccounting for 43 wt % or more of the resultant sugars. Furthermore, itacts on a sucrose solution under the same conditions and producesnystose accounting for 13 wt % or less of the resultant sugars. In theprocesses described in Japanese Patent Publication No. 62184/1988 andJapanese Patent Laid-Open Publication No. 268190/1986, the rates ofconversion from sucrose to 1-kestose are only 36 to 41% at maximum. Inthese processes, 11 to 23% of nystose is produced along with 1-kestose.The process according to the present invention has a marked advantageover the prior art described in the publications in selectivelyproducing 1-kestose.

In the present invention, the enzyme is allowed to react on sucrose toproduce 1-kestose. In a preferred embodiment of the present invention, asucrose solution with a concentration of 50 wt % or higher, preferably50 wt % to 60 wt %, is allowed to react at pH 5.0 to 10.0, preferably atpH 5.5 to 10.0, and a temperature of 50 to 65° C., preferably 50 to 60°C. Furthermore, in a preferred embodiment of the present invention, theenzyme having fructose transferase activity is allowed to react withsucrose solution at a rate of 0.5 to 100 units, preferably 3 to 100units, per 1 g of sucrose and a temperature of 50 to 65° C., preferably50 to 60° C., for 2 to 100 hours, preferably for 2 to 40 hours.

It is preferable that the enzyme be deactivated after the transferreaction is over, preferably by adding activated carbon (e.g., Taikoactivated carbon S-W50) at a rate of 0.1 to 1.0 wt % of the solidscontent and heating the reacting solution at a temperature of 90 to 95°C. for 20 minutes to 60 minutes, concurrently with the decoloration ofthe reacting solution.

In a preferred embodiment of the present invention, the resultant sugarscontain 43 wt % or more of 1-kestose and 13 wt % or less of nystose. Thecomposition can be improved under more preferable conditions to 44 wt %or more of 1-kestose and 7 wt % or less of nystose.

Step (i)(2)

The enzyme for use in step (i)(2) is an enzyme derived from aPenicillium species which can act on sucrose to produce 1-kestoseaccounting for 46 wt % or more of the resultant sugars. Enzymes havingfructose transferase activity which are used preferably in the presentinvention include the novel enzyme described below:

The enzyme is prepared from a Penicillium microorganism, preferably fromPenicillium roqueforti, specifically Penicillium roqueforti IAM7254strain.

The enzyme can be recovered from a culture by incubating themicroorganism in a suitable medium (e.g., a medium containing 5 to 30%of sucrose, 1 to 10% of corn steep liquor, 0.05 to 0.3% of urea, 0.2 to3.0 of potassium dihydrogenphosphate, and 0.01 to 0.1% of magnesiumsulfate heptahydrate), at initial pH of 6.5 to 7.5 and a temperature of25 to 30° C. for 2 to 5 days.

It is preferable that the enzyme be purified by a known purificationmethod. Preferable purification methods include salting out using a saltsuch as ammonium sulfate; precipitation using an organic solvent such asmethanol; ethanol or acetone, adsorption using starch; ultrafiltration;gel filtration chromatography; ion exchange chromatography; and variousother chromatographic procedures.

The novel enzyme showed the following properties:

Activity

The enzyme cuts the β-D-fructofuranoside bond of a sugar having aβ-D-fructofuranoside bond, such as sucrose, 1-kestose or raffinose, andtransfers the resultant fructosyl group specifically to the C-1 position(hydroxyl group) of the terminal fructosyl group of sugars.

The enzyme also acts on sucrose solution with a concentration of 50 wt %or higher at pH 6.0 to 9.0 and a temperature of 35 to 55° C. to produce1-kestose accounting for 46 wt % or more of the resultant sugars.

The enzyme can react with a sucrose solution with a concentration of 50wt % or higher at a rate of 0.5 to 100 units per 1 g of sucrose, at pH6.0 to 9.0 and a temperature of 35 to 55° C. for 2 to 100 hours, toproduce 1-kestose accounting for 46 wt % or more of the resultantsugars. The enzyme also acts on a sucrose solution under the sameconditions to produce nystose accounting for 7 wt % or less of theresultant sugars. In this sense, the enzyme is significantly suitable inselectively producing 1-kestose.

Substrate Specificity

The enzyme effectively acts on sucrose, 1-kestose, raffinose, but not onturanose, maltose.

Optimum Temperature

The optimum temperature of the enzyme is 40 to 50° C.

Stability to Temperature

The enzyme retains at least 60% of relative activity at pH 7.0 and 55°C. or less after 30 minutes.

Optimum pH

The optimum pH of the enzyme is 6.0 to 7.0.

Stable pH

The enzyme is remarkably stable in the range of pH 4.0 to 8.0, retainingat least 90% of relative activity at 40° C. after 30 minutes.

Molecular Weight

The molecular weight of the enzyme as measured by gel filtrationchromatography is 315,000.

Inhibition of Activity

The enzyme is inhibited by glucose, which is a byproduct of the fructosetransfer reaction.

Enzyme Kinetics

The enzyme's km is 1.1 M.

In step (i)(2) of the present invention, the enzyme is allowed to reacton sucrose to produce 1-kestose. According to a preferred embodiment ofthe present invention, a sucrose solution with a concentration 50 wt %or higher, preferably 55 to 65 wt %, is allowed to react at pH 6.0 to9.0, preferably 6.0 to 8.0, and a temperature of 35 to 55° C.,preferably 40 to 50° C. Further, according to a preferred embodiment ofthe present invention, the enzyme having fructose transferase activityis added to a sucrose solution at a rate of 0.5 to 100 units, preferably2 to 50 units, per 1 g of sucrose to react at a temperature of 35 to 55°C., preferably 40 to 50° C., for 2 to 100 hours, preferably 4 to 100hours.

It is preferable that the enzyme be deactivated after the transferreaction is over, preferably by adding activated carbon (e.g., Taikoactivated carbon S-W50) at a rate of 0.1 to 1.0 wt % of the solidscontent and heating the reacting solution at a temperature of 40 to 70°C. for 20 minutes to 60 minutes, concurrently with the decoloration ofthe reacting solution.

According to a preferred embodiment of the present invention, theresultant sugars contain 46 wt % or more of 1-kestose and 7 wt % or lessof nystose.

Step (i)(3)

The enzyme for use in step (i)(3) is an enzyme derived from aScopulariopsis species which can act on sucrose to produce 1-kestoseaccounting for 53 wt % or more of the resultant sugars. Enzymes havingfructose transferase activity which are used preferably in the presentinvention include the novel enzyme described below:

The enzyme is prepared from a Scopulariopsis microorganism, preferablyfrom Scopulariopsis brevicaulis, specifically Scopulariopsis brevicaulisIF04843 strain.

The enzyme can be recovered from a culture by incubating themicroorganism in a suitable medium (e.g., a medium containing 3.0 to10.0% of sucrose, 5 to 15% of corn steep liquor, 0.05 to 0.3% of urea,0.2 to 3.0 of potassium dihydrogenphosphate, and 0.01 to 0.1% ofmagnesium sulfate heptahydrate), at initial pH of 6.5 to 7.5 and atemperature of 20 to 30° C. for 3 to 8 days. The enzyme is preparedpreferably by recovering the fungus body from the culture using acentrifuge such as a basket type; suspending the recovered fungus bodyin a buffer at pH 7.0; obtaining a crude enzyme suspension by anultrasonic treatment and a membrane treatment; then treating with anionexchange, gel filtration, chromatographic focusing, or other procedureon the crude enzyme suspension.

The novel enzyme showed the following properties:

Activity

The enzyme cuts the β-D-fructofuranoside bond of a sugar having aβ-D-fructofuranoside bond, such as sucrose, 1-kestose or raffinose, andtransfers the resultant fructosyl group specifically to the C-1 position(hydroxyl group) of the terminal fructosyl group of sugars.

The enzyme also acts on a sucrose solution with a concentration of 50 wt% or higher at pH 6.0 to 10.0 and a temperature of 35 to 50° C. toproduce 1-kestose accounting for 53 wt % or more of the resultantsugars.

The enzyme can react with a sucrose solution with a concentration of 50wt % or higher, preferably 50% to 55 wt % at pH 6.0 to 10.0, preferably7.0 to 9.8, and a temperature of 35 to 50° C., preferably 35 to 40° C.,to produce 1-kestose accounting for 53 wt % or more of the resultantsugars. Preferably, the enzyme is allowed to react at a rate of 0.5 to100 units, more preferably 2 to 20 units, per 1 g of sucrose. The enzymeaccording to the present invention also acts on a sucrose solution underthe same conditions to produce nystose accounting for 5 wt % or less ofthe resultant sugars. In this sense, the enzyme is significantlysuitable in selectively producing 1-kestose.

Substrate Specificity

The enzyme effectively acts on sucrose, 1-kestose, raffinose, but not onturanose, maltose.

Optimum Temperature

The optimum temperature of the enzyme is 40° C.

Optimum pH

The optimum pH of the enzyme is 7.0.

Stable pH

The enzyme is remarkably stable in the range of pH 6.0 to 10.0,retaining at least 80% of relative activity at 40° C. after 30 minutes.

Molecular Weight

The molecular weight of the enzyme as measured by gel filtrationchromatography is 360 to 380 kDa.

An analysis by SDS-PAGE has revealed that the enzyme has a molecularweight of about 54 kDa and comprises a subunit with a molecular weightof about 51 kDa with the glycoside chain removed.

Isoelectric Point

The isoelectric point as measured by two dimensional electrophoresis andhydrophobic chromatography is about 3.8 to 3.9.

Inhibition of Activity

The enzyme is inhibited by glucose, which is a byproduct of the fructosetransfer reaction.

Enzyme Kinetics

The enzyme's km is 0.75 M. The enzyme is uncompetitively inhibited byglucose with an inhibition constant (Ki) of 0.125 M.

In step (i)(3) of the present invention, the enzyme is allowed to reacton sucrose to produce 1-kestose. According to a preferred embodiment ofthe present invention, a sucrose solution with a concentration 50 wt %or higher, preferably 50 to 55 wt %, is allowed to react at pH 6.0 to10.0, preferably 7.0 to 9.8, and a temperature of 35 to 50° C.,preferably 35 to 40° C. Further, according to a preferred embodiment ofthe present invention, the enzyme having fructose transferase activityis added to a sucrose solution at a rate of 0.5 to 100 units, preferably2 to 20 units, per 1 g of sucrose to react at a temperature of 35 to 50°C., preferably 35 to 40° C., for 2 to 100 hours, preferably 9 to 60hours.

It is preferable that the enzyme be deactivated after the transferreaction is over, preferably by adding activated carbon (e.g., Taikoactivated carbon S-W50) at a rate of 0.1 to 1.0 wt % of the solidscontent and heating the reacting solution at a temperature of 90 to 95°C. for 20 minutes to 60 minutes, concurrently with the decoloration ofthe reacting solution.

According to a preferred embodiment of the present invention, theresultant sugars contain 53 wt % or more of 1-kestose and 5 wt % or lessof nystose. The composition can be improved under more preferableconditions to 55 wt % or more of 1-kestose and 4 wt % or less ofnystose.

According to a preferred embodiment of the present invention, it isdesired that the solution containing 1-kestose which has been obtainedin either of steps (i)(1) through (3) above be filtered to remove fungusbody, etc., and decolorized and deodorized by using activated carbon,prior to the subsequent step (ii). Decoloration and deodorization may beachieved by adding activated carbon accounting for about 0.1 to 1.0 wt %of the solids content in the 1-kestose solution and stirring at atemperature of 40 to 70° C. for 20 to 60 minutes. It is preferable thatthe solution be further desalted with cation and anion exchange resin todesalt and filtered again to provide a colorless and transparent1-kestose solution.

Step (ii): Chromatographic Separation

The reacting solution containing 1-kestose, which has been obtainedusing the above enzyme, is chromatographically separated to preparecrystal 1-kestose. A preferred method of chromatographic separation issimulated-moving bed chromatographic separation, which is generally usedfor producing isomerized sugars.

The chromatographic separation provides a fraction containing 1-kestoseat a purity of 80% or higher, preferably 90% or higher.

In the process according to the present invention, the relative contentof sugars of which molecular weights are higher than that of 1-kestose,such as nystose, is low as already explained. Therefore, 1-kestose caneffectively be purified by a single procedure of two-componentsimulated-moving bed chromatographic separation using the enzyme andreacting conditions as described in step (i), Crn. while two or morerepeated operations are required in the prior art. A preferredembodiment of the present invention enables 1-kestose to be purified to80 to 95% by the this procedure.

In this step, a fraction containing 1-kestose at a purity of 80%,preferably 90% or higher, is obtained. It is preferable that the highlypure 1-kestose solution be separated to minimize the proportion ofnystose in the remaining sugar content, as a lower nystose content wouldresult in larger crystals of 1-kestose. It is desired that the nystosecontent be 10 wt % or less of 1-kestose.

According to a preferred embodiment of the present invention, it ispreferable that the obtained fraction be treated with activated carbonand desalted before proceeding to the subsequent step (iii). Treatingwith activated carbon and desalting may be performed in the same manneras in step (i).

Step (iii): Crystallization

In the third aspect of the present invention, step (iii) is acrystallizing step. The procedure for step (iii) may preferably be thesame as in the first or second aspect of the present invention.Similarly, the preferred embodiment of the first and second aspects ofthe present invention may also be applicable as the preferred embodimentof the third aspect of the present invention.

Crystal 1-Kestose

According to the preferred embodiments of the present invention,granular crystal 1-kestose having grain sizes of 0.3 to 2 mm, preferably0.5 to 1.2 mm can be obtained. Unlike powdery crystals, large crystalsof 1-kestose do not easily coagulate while drying, making it possible toobtain a product of a uniform grain size. Furthermore, the crystal1-kestose according to the present invention has high fluidity and,therefore, is less likely to dust or consolidate. Advantages expectedfrom this characteristic include little deviation in small packages suchas for table sugar, and little classification when mixed with granulatedsugar.

EXAMPLES

The present invention is further illustrated by the following exampleswhich are not intended as a limitation of the invention. Figures inpercentage hereafter refer to wt % unless otherwise specified.

In the following examples, the crystallizing sample used was prepared byprocedures such as chromatographically separating and purifying from acommercially available mixture of fructooligosaccharides, Meioligo G orMeioligo P (Meiji Seika), or chromatographically separating andpurifying from a sucrose solution which had been allowed to react withfructose transferase as described in the following examples.

In the following examples, the sugar composition in the crystallizingsample varies, reflecting the difference in the separating conditions.

Example A Example A1

A crystallizing sample with a sugar composition of 92% of 1-kestose, 5%of sucrose, and 3% of nystose, which had been prepared according toExample C1 as described later, was used.

The crystallizing sample was concentrated to a Brix of 80. Then, afterheating the concentrate to a temperature of 75° C., seed crystals of1-kestose accounting for 1% of the solids content in the concentratewere added. Crystals were allowed to grow for 0.5 hour. The solution wasthen concentrated under a reduced pressure at an absolute pressure of 80mmHg for 30 minutes. Through the concentration procedure, thetemperature of the solution dropped to 55° C., and the Brix became 83.As microcrystals of 1-kestose were generated during the concentrationprocedure, the concentrate was heated at 75° C. and maintained at thetemperature for 1 hour to dissolve the microcrystals. As a result, theBrix of the portion of the concentrate except the crystals becameapproximately 80.

The crystal growing step and the microcrystal dissolving step above wererepeated four times to allow 1-kestose to crystallize sufficiently.Then, the crystals were recovered by centrifugation at room temperature,and dried to finally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 60%.

A microscopic examination revealed that the majority of the crystalsranged from 0.3 to 2 mm in length, and that there was no crystalcoagulation.

The grain size distribution of the crystals was investigated byscreening the crystals through 20, 40 and 60 mesh automatic sieves for 5minutes. The result is shown in the table below. The prevailing crystalsize was found to be 0.5 mm to 1.2 mm in length. It should be noted thatthe actual lengths of the oversize crystals exceeded the diagonal lengthof the mesh, considering that the crystals of 1-kestose have rectangularshapes.

TABLE 1 Mesh Side length Diagonal length oversize 20 0.84 1.19 0.5%75.9% 40 0.37 0.52 18.1% 60 0.25 0.35 5.5%

Example A2

A crystallizing sample with a sugar composition of 95% of 1-kestose, 4%of sucrose, and 1% of nystose, which had been prepared according toExample D2 as described later, was used.

The crystallizing sample was concentrated to a Brix of 80. Then, afterheating the concentrate to a temperature of 80° C., seed crystals of1-kestose accounting for 0.5% of the solids content in the concentratewere added. Crystals were allowed to grow for 0.5 hour. The solution wasthen concentrated under a reduced pressure at an absolute pressure of160 mmHg for 30 minutes. Through the concentration procedure, thetemperature of the solution dropped to 70° C., and the Brix became 83.As microcrystals of 1-kestose were generated during the concentrationprocedure, the concentrate was heated at 95° C. and maintained at thetemperature for 1 hour to dissolve the microcrystals.

The crystal growing step and the microcrystal dissolving step above wererepeated four times. As microcrystals failed to dissolve completely inthe fourth microcrystal dissolving step, water accounting for about 2%of the solids content in the concentrate was added to dissolve themicrocrystals. Then, the crystal growing step and the microcrystaldissolving step above were repeated two more times. Then, the crystalswere recovered by centrifugation at room temperature, and dried tofinally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 57%. A microscopic examination revealed thatthe a majority of the crystals were about 1 mm in length.

Example A3

A crystallizing sample with a sugar composition of 95% of 1-kestose, 4%of sucrose, and 1% of nystose, which had been prepared according toExample D2 as described later, was used.

The crystallizing sample was concentrated to a Brix of 75. Then, afterheating the concentrate to a temperature of 70° C., the solution wasconcentrated at an absolute pressure of 80 mmHg for 15 minutes toproduce microcrystals. With the microcrystals as nuclei, crystals wereallowed to grow at 70° C. for 6.0 hours. The solution was thenconcentrated under a reduced pressure at an absolute pressure of 80 mmHgfor 30 minutes. Through the concentration procedure, the temperature ofthe solution dropped to 55° C. As microcrystals of 1-kestose weregenerated during the concentration procedure, the concentrate was heatedat 75° C. and maintained at the temperature for 1 hour to dissolve themicrocrystals.

The crystal growing step and the microcrystal dissolving step above wererepeated four times to allow 1-kestose to crystallize sufficiently.Then, the crystals were recovered by centrifugation at room temperature,and dried to finally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 59%. A microscopic examination revealed thatthe a majority of the crystals were about 1 mm in length.

The grain size distribution of the crystals was investigated byscreening the crystals through 20, 24, 32 and 42 mesh sieves.

The result is shown in the table below. The prevailing crystal size wasfound to be 0.7 mm to 1.2 mm in length. It should be noted that theactual lengths of the oversize crystals exceeded the diagonal length ofthe mesh, considering that the crystals of 1-kestose have rectangularshapes.

TABLE 2 Mesh Side length Diagonal length oversize 20 0.84 1.19 6.5%35.8% 24 0.71 1.00 41.7% 32 0.50 0.70 13.8% 42 0.35 0.49 2.2%

Example A4

A crystallizing sample with a sugar composition of 92% of 1-kestose, 2%of sucrose, and 6% of nystose was prepared from Meioligo P bychromatographic separation using a two-component simulated-moving bedchromatographic separator.

The crystallizing sample was concentrated to a Brix of 80. Then, afterheating the concentrate to a temperature of 70° C., the solution wasconcentrated at an absolute pressure of 80 mmHg for 10 minutes toproduce microcrystals. With the microcrystals as nuclei, crystals wereallowed to grow at 70° C. for 3 hours. The solution was thenconcentrated under a reduced pressure at an absolute pressure of 80 mmHgfor 30 minutes. Through the concentration procedure, the temperature ofthe solution cooled to 55° C. As microcrystals of 1-kestose weregenerated during the concentration procedure, the concentrate was heatedat 75° C. and maintained at the temperature for 1 hour to dissolve themicrocrystals.

The crystal growing step and the microcrystal dissolving step above wererepeated three times. The crystallizing sample was added to make up forthe loss in volume caused in the concentration procedure. Then, thecrystal growing step and the microcrystal dissolving step above wererepeated two more times. Then, the crystals were recovered bycentrifugation at room temperature, and dried to finally make 1-kestosecrystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 60%. A microscopic examination revealed thatthe a majority of the crystals ranged approximately from 0.3 to 1 mm inlength.

Example A5

A crystallizing sample with a sugar composition of 95% of 1-kestose, 4%of sucrose, and 1% of nystose, which had been prepared according toExample D2 as described later, was used.

The crystallizing sample was concentrated to a Brix of 85. Then, afterheating the concentrate to a temperature of 80° C., seed crystals of1-kestose accounting for 0.5% of the solids content in the concentratewere added. Crystals were allowed to grow for 30 minutes. After thesolution was cooled to 70° C., taking 30 minutes, crystals were allowedto grow at 70° C. for 30 minutes. Further, after the solution was cooledto 60° C., taking 40 minutes, crystals were allowed to grow at 60° C.for 30 minutes. Furthermore, after the solution was cooled to 50° C.,taking 60 minutes, crystals were allowed to grow at 50° C. for 30minutes.

Then, the crystals were recovered by centrifugation at room temperature,and dried to finally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 98%, and the yield ofcrystals was approximately 45%. A microscopic examination revealed thatthe a majority of the crystals ranged approximately from 0.3 to 2 mm inlength.

Example A6

A crystallizing sample with a sugar composition of 95% of 1-kestose, 4%of sucrose, and 1% of nystose, which had been prepared according toExample D2 as described later, was used.

The crystallizing sample was concentrated to a Brix of 85. Then, theconcentrate was heated gradually from 20° C. to 80° C. to producemicrocrystals at around 50 to 60° C. With the microcrystals as nuclei,crystals were allowed to grow at 80° C. for 30 minutes. After thesolution was cooled to 70° C., taking 30 minutes, crystals were allowedto grow at 70° C. for 30 minutes. Further, after the solution was cooledto 60° C., taking 40 minutes, crystals were allowed to grow at 60° C.for 30 minutes. Furthermore, after the solution was cooled to 50° C.,taking 60 minutes, crystals were allowed to grow at 50° C. for 30minutes.

Then, the crystals were recovered by centrifugation at room temperature,and dried to finally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 43%. A microscopic examination revealed thatthe majority of the crystals ranged approximately from 0.3 to 2 mm inlength.

Example A7

A crystallizing sample with a sugar composition of 95% of 1-kestose, 4%of sucrose, and 1% of nystose, which had been prepared according toExample D2 as described later, was used.

The crystallizing sample was crystallized in a continuouscrystallization procedure.

The crystallizing sample was concentrated to a Brix of 85. Seed crystalsof 1-kestose accounting for 0.1% of the solids content in theconcentrate were added. Crystals were allowed to grow at 80° C. for 30minutes. After the solution was cooled to 70° C., taking 30 minutes,crystals were allowed to grow at 70° C. for 30 minutes. Further, afterthe solution was cooled to 60° C., taking 40 minutes, crystals wereallowed to grow at 60° C. for 30 minutes. Furthermore, after thesolution was cooled to 50° C., taking 60 minutes, crystals were allowedto grow at 50° C. for 30 minutes. Finally, after the solution was cooledto 30° C., taking 120 minutes, crystals were allowed to grow at 30° C.for 6 hours. The crystals were recovered by centrifugation at roomtemperature, and dried to finally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 51%.

As the purity of 1-kestose in the molasses was 92%, it was concentratedto a Brix of 84, and crystallized in the same procedure. The purity ofthe 1-kestose crystals obtained was 97%, and the yield of crystals wasapproximately 44%.

The total yield of 1-kestose so far was 72%.

Then, as the purity of 1-kestose in the molasses was 89%, it wasconcentrated to a Brix of 84, and crystallized in the same procedure.The purity of the 1-kestose crystals obtained was 95%, and the yield ofcrystals was approximately 55%. The total yield of 1-kestose so far was85%.

As the purity of 1-kestose in the molasses was 79%, it was concentratedto a Brix of 84, and crystallized in the same procedure. However,separation at room temperature was impracticable due to extensivedeposition of microcrystals.

Example A8

A crystallizing sample with a sugar composition of 95% of 1-kestose, 4%of sucrose, and 1% of nystose, which had been prepared according toExample D2 as described later, was used.

The crystallizing sample was concentrated to a Brix of 85. Then, afterheating the concentrate to a temperature of 80° C., seed crystals of1-kestose accounting for 0.5% of the solids content in the concentratewere added. Crystals were allowed to grow at 80° C. for 15 hours. Afterthe solution was cooled to 60° C., taking 2 hours, crystals were allowedto grow at 60° C. for 5 hours. Furthermore, after the solution wascooled to 40° C., taking 4 hours, crystals were allowed to grow at 40°C. for 48 hours. The crystals were recovered by centrifugation at roomtemperature, and dried to finally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 50%. A microscopic examination revealed thatthe majority of the crystals ranged approximately from 0.3 to 2 mm inlength.

The grain size distribution of the crystals was investigated byscreening the crystals through 20, 24, 32 and 42 mesh sieves.

The result is shown in the table below. The prevailing crystal size wasfound to be 1.0 mm to 1.2 mm in length. It should be noted that theactual lengths of the oversize crystals exceeded the diagonal length ofthe mesh, considering that the crystals of 1-kestose have rectangularshapes.

TABLE 3 Mesh Side length Diagonal length oversize 20 0.84 1.19 7.3%68.5% 24 0.71 1.00 16.0% 32 0.50 0.70 6.4% 42 0.35 0.49 1.8%

Comparative Example

The crystal 1-kestose prepared in Example A above was mixed with sucroseand examined for the properties of the mixture.

The crystal 1-kestose prepared in Example A2 and granulated sugar HA(Nippon Beet Sugar) were screened separately. The 40 mesh oversizeportion of each was mixed together at a rate of 80% of sucrose and 20%of 1-kestose and used as a coarse (40 mesh oversize) mixture sample ofsucrose and 1-kestose.

In addition, crystal 1-kestose prepared in Example A2 above andgranulated sugar HA (Nippon Beet Sugar) were separately crushed with atable grinder and screened. The 100 mesh undersize portion of each wasmixed together at a rate of 80% of sucrose and 20% of 1-kestose and usedas a fine mixture sample of sucrose and 1-kestose.

These samples were analyzed using a powder tester (Hosokawa Micron) forthe items listed in the table below according to standard procedures.The results are shown in the table below:

TABLE 4 Fine Coarse Item sample sample Fluidity Angle of repose 49 39Spatula angle 65 40 Compressibility 49 13 Dispersibility 27 11 Variationas packed Poor Good

These results show that the coarse sample had superior fluidity and lessvariation as packed. The fine sample was found to consolidate whencompressed.

Example B Example B1

(1) A 15 L of medium containing 5% of sucrose, 6.5% of yeast extract,and 0.5% of CMC(caroboxymetyl cellulose) was placed in a 30 L jarfermenter, adjusted to pH 6.5, and sterilized at 120° C. for 30 minutes.Asperaillus niger ATCC20611 was inoculated in a medium placed in a 1 LErlenmeyer flask and containing 5.0% of sucrose, 2.0% of powderedbouillon, and 0.5% of CMC and incubated at 28° C. for 21 hours, thenimplanted in the above medium and incubated at 28° C. for 96 hours.After incubation was over, fungus body was recovered from the culture,using a basket type centrifuge, and freeze-dried to make dried fungusbody for use as enzyme in the following reactions.

The fructose transferase activity of the dried fungus body wasapproximately 14,000 units per 1 g of dried fungus body.

(2) 10 L of sucrose solution adjusted to a concentration of 60%, pH10.0, and a temperature of 60° C. was placed in a reaction chamber. Theenzyme which had been prepared in (1) above was added to the reactionchamber at a rate of 60 units per 1 g of sucrose, and allowed to reactat a temperature of 60° C. and pH 10.0 for 40 hours. Then, activatedcarbon (Taiko activated carbon S-W50) was added at a rate of 1.0% of thesolids content to deactivate the enzyme and decolorize the solution at atemperature of 95° C. for 30 minutes.

The sugar composition of the deactivated solution was 44% of 1-kestose,32% of sucrose, 17% of monosaccharides, and 7% of nystose.

The deactivated solution was filtered to remove any impurities such asthe fungus body and the activated carbon. Then, activated carbon (Taikoactivated carbon S-W50) was added to the filtrate at a rate of 0.5% ofthe solids content, and stirred at a temperature of 60° C. for 20minutes to decolorize again. After filtrating to remove the activatedcarbon in the same manner as above, the solution was desalted throughcation and anion exchange resin, then filtrated again to make acolorless and transparent 1-kestose solution (chromatographic sample).In the decolorizing and desalting procedure, the decomposition of1-kestose was hardly observed.

In order to improve the purity of 1-kestose in the chromatographicsample obtained above, the solution was chromatographically separated bya two-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 92% of 1-kestose,3% of sucrose, and 5% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).

Example B2

Enzyme which had been prepared in the same manner as in Example B1 (1)was added to 10 L of sucrose solution adjusted to a concentration of50%, pH 9.0, and a temperature of 50° C. at a rate of 100 units per 1 gof sucrose, and allowed to react at a temperature of 50° C. and pH 9.0for 2 hours. Then, activated carbon (Taiko activated carbon S-W50) wasadded at a rate of 1.0% of the solids content to deactivate the enzymeand decolorize the solution at a temperature of 95° C. for 30 minutes.

The sugar composition of the deactivated solution was 43% of 1-kestose,30% of sucrose, 18% of monosaccharides, and 9% of nystose.

The deactivated solution was filtered to remove any impurities such asthe fungus body and the activated carbon. Then, activated carbon (Taikoactivated carbon S-W50) was added to the filtrate at a rate of 0.5% persolids content, and stirred at a temperature of 60° C. for 20 minutes todecolorize again. After filtrating to remove the activated carbon in thesame manner as above, the solution was desalted through cation and anionexchange resin, then filtrated again to make a colorless and transparent1-kestose solution (chromatographic sample). In the decolorizing anddesalting procedure, the decomposition of 1-kestose was hardly observed.

In order to improve the purity of 1-kestose in the chromatographicsample obtained above, the solution was chromatographically separated bya two-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 90% of 1-kestose,3% of sucrose, and 7% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).

Example B3

Enzyme which had been prepared in the same manner as in Example B1 (1)was added to 10 L of sucrose solution adjusted to a concentration of60%, pH 5.5, and a temperature of 60° C. at a rate of 3.0 units per 1 gof sucrose, and allowed to react at a temperature of 60° C. and pH 5.5for 4 hours. Then, activated carbon (Taiko activated carbon S-W50) wasadded at a rate of 0.3% of the solids content to deactivate the enzymeand decolorize the solution at a temperature of 95° C. for 30 minutes.

The sugar composition of the deactivated solution was 43% of 1-kestose,22% of sucrose, 22% of monosaccharides, and 13% of nystose.

The deactivated solution was filtered to remove any impurities such asthe fungus body and the activated carbon. Then, activated carbon (Taikoactivated carbon S-W50) was added to the filtrate at a rate of 0.2% persolids content, and stirred at a temperature of 60° C. for 20 minutes todecolorize again. After filtrating to remove the activated carbon in thesame manner as above, the solution was desalted through cation and anionexchange resin, then filtrated again to make a colorless and transparent1-kestose solution (chromatographic sample). In the decolorizing anddesalting procedure, the decomposition of 1-kestose was hardly observed.

In order to improve the purity of 1-kestose in the 1-kestose solutionobtained above, the solution was chromatographically separated by atwo-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 80% of 1-kestose,7% of sucrose, and 13% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).

Example B4

The crystallizing sample with a sugar composition of 90% of 1-kestose,3% of sucrose, and 7% of nystose, which had been prepared according tothe procedure as described in Example B2, was concentrated to a Brix of80. After heating the concentrate to a temperature of 70° C., thesolution was concentrated at an absolute pressure of 80 mmHg for 10minutes to produce microcrystals. With the microcrystals as nuclei,crystals were allowed to grow at 70° C. for 3 hours. The solution wasconcentrated under a reduced pressure at an absolute pressure of 80 mmHgfor 30 minutes. Through the concentration procedure, the temperature ofthe solution dropped to 55° C. As microcrystals of 1-kestose weregenerated during the concentration procedure, the concentrate was heatedat 75° C. and maintained at the temperature for 1 hour to dissolve themicrocrystals.

The crystal growing step and the microcrystal dissolving step above wererepeated three times. The crystallizing sample was added to make up forthe loss in volume caused in the concentration procedure. Then, thecrystal growing step and the microcrystal dissolving step above wererepeated two more times. Then, the crystals were recovered bycentrifugation at room temperature, and dried to finally make 1-kestosecrystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 60%.

A microscopic examination revealed that the majority of the crystalsranged approximately from 0.3 to 1 mm in length.

Example B5

A crystallizing sample with a sugar composition of 92% of 1-kestose, 3%of sucrose, and 5% of nystose, which had been prepared in Example B1,was crystallized in a continuous crystallization procedure.

The crystallizing sample was concentrated to a Brix of 85. Seed crystalsof 1-kestose accounting for 0.1% of the solids content in theconcentrate were added. Crystals were allowed to grow at 80° C. for 30minutes. After the solution was cooled to 70° C., taking 30 minutes,crystals were allowed to grow at 70° C. for 30 minutes. Further, afterthe solution was cooled to 60° C., taking 40 minutes, crystals wereallowed to grow at 60° C. for 30 minutes. Furthermore, after thesolution was cooled to 50° C., taking 60 minutes, crystals were allowedto grow at 50° C. for 30 minutes. Finally, after the solution was cooledto 30° C., taking 120 minutes, crystals were allowed to grow at 30° C.for 6 hours. The crystals were recovered by centrifugation at roomtemperature, and dried to finally make 1-kestose crystals.

The purity of the 1-kestose crystals obtained was 99%, and the yield ofcrystals was approximately 51%.

As the purity of 1-kestose in the molasses was 85%, it was concentratedto a Brix of 84, and crystallized in the same procedure as above. Thepurity of the 1-kestose crystals obtained was 95%, and the yield ofcrystals was approximately 55%.

The total yield of 1-kestose so far was 78%.

Then, as the purity of 1-kestose in the molasses was 74%, it wasconcentrated to a Brix of 84, and crystallized in the same procedure asabove. However, separation at room temperature was impracticable due toextensive deposition of microcrystals.

Example C Example C1

(1) A 15 L of medium containing 22.5% of sucrose, 1.5% of yeast extract,and 3.0% of corn steep liquor was placed in a 30 L jar fermenter,adjusted to pH 6.8, and sterilized at 120° C. for 30 minutes.Penicillium roqueforti (IAM7254 strain) was inoculated in a mediumplaced in a 1 L Erlenmeyer flask and containing 15.0% of sucrose, 1.0%of yeast extract, and 2.0% of corn steep liquor and incubated at 28° C.for three days, then implanted in the above medium and incubated at 28°C. for four days. After incubation was over, fungus body was recoveredfrom the culture, using a basket type centrifuge, and freeze-dried tomake dried fungus body for use as enzyme in the following reactions.

The fructose transferase activity of the dried fungus body wasapproximately 95 to 135 units per 1 g of dried fungus body.

(2) Enzyme which had been prepared as described above was added to 10 Lof sucrose solution adjusted to a concentration of 55 wt %, pH 7.0, anda temperature of 50° C., at a rate of 10 units per 1 g of sucrose, andallowed to react at a temperature of 50° C. and pH 7.0 for 4 hours.Then, activated carbon (Taiko activated carbon S-W50) was added at arate of 0.3% of the solids content to deactivate the enzyme anddecolorize the solution at a temperature of 95° C. for 30 minutes. Thesugar composition of the solution was 46% of 1-kestose, 18% of sucrose,30% of monosaccharides, and 6% of nystose. The deactivated solution wasfiltered to remove any impurities such as the fungus body. Then, thefiltrate was decolorized and desalted through activated carbon and ionexchange resin, then filtrated again to make a colorless and transparent1-kestose solution.

In order to improve the purity of 1-kestose in the 1-kestose solutionobtained above, the solution was chromatographically separated by atwo-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 92% of 1-kestose,5% of sucrose, and 3% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).

Example C2

Enzyme which had been prepared as described in Example C1 (1) was addedto 10 L of sucrose solution adjusted to a concentration of 65 wt %, pH8.0, and a temperature of 40° C., at a rate of 50 units per 1 g ofsucrose, and allowed to react at a temperature of 40° C. and pH 8.0 for5 hours. Then, activated carbon (Taiko activated carbon S-W50) was addedat a rate of 1.0% of the solids content to deactivate the enzyme anddecolorize the solution at a temperature of 95° C. for 30 minutes.

The sugar composition of the solution was 47% of 1-kestose, 18% ofsucrose, 28% of monosaccharides, and 7% of nystose.

The deactivated solution was filtered to remove any impurities such asthe fungus body. Then, the filtrate was decolorized and desalted throughactivated carbon and ion exchange resin, then filtrated again to make acolorless and transparent 1-kestose solution (chromatographic sample).

In order to improve the purity of 1-kestose in the 1-kestose solutionobtained above, the solution was chromatographically separated by atwo-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 90% of 1-kestose,3% of sucrose, and 7% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).The crystallizing sample thus prepared was treated in the same procedureas in Example B4 described above to finally make similar crystal1-kestose.

Example C3

Enzyme which had been prepared as described in Example C1 (1) was addedto 10 L of sucrose solution adjusted to a concentration of 65 wt %, pH6.0, and a temperature of 40° C., at a rate of 2 units per 1 g ofsucrose, and allowed to react at a temperature of 40° C. and pH 6.0 for100 hours. Then, activated carbon (Taiko activated carbon S-W50) wasadded at a rate of 1.0% of the solids content to deactivate the enzymeand decolorize the solution at a temperature of 95° C. for 30 minutes.

The sugar composition of the solution was 46% of 1-kestose, 19% ofsucrose, 28% of monosaccharides, and 7% of nystose.

The deactivated solution was filtered to remove any impurities such asthe fungus body. Then, the filtrate was decolorized and desalted throughactivated carbon and ion exchange resin, then filtrated again to make acolorless and transparent 1-kestose solution (chromatographic sample).

In order to improve the purity of 1-kestose in the 1-kestose solutionobtained above, the solution was chromatographically separated by atwo-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 80% of 1-kestose,7% of sucrose, and 13% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).

Example D Example D1

(1) Purification of Enzyme

A 15 L of medium containing 5.0% of sucrose, 10.5% of corn steep liquor,and 0.1% of urea was placed in a 30L jar fermenter, adjusted to pH 7.0,and sterilized at 120° C. for 30 minutes. Scopulariopsis brevicaulisIFO4843 strain was inoculated in a medium placed in a 1 L Erlenmeyerflask and containing 5.0% of sucrose, 1.0% of yeast extract, and 2.0% ofcorn steep liquor and incubated at 28° C. for 29 hours, then implantedas fungus seeds and incubated at 28° C. for seven days. After incubationwas over, fungus body was recovered from the culture, using a baskettype centrifuge, and freeze-dried to make dried fungus body.

The fructose transferase activity of the dried fungus body wasapproximately 5.2 to 8.3 units per 1 g of dried fungus body.

The freeze-dried fungus body powder prepared above was suspended in aMcIlvaine buffer (pH 7.0), and ultrasonically crushed. The crushed cellswere ultrafiltrated through a membrane filter (cutoff molecular weight:100,000). The supernatant obtained as crude enzyme was separated throughan ion exchange column (Q-Sepharose column 60/100, Pharmacia), which hadbeen equilibrated with Tris-HCl buffer (pH 7.3), at an NaCl gradient of0 to 1 M. Two fractions which exhibited enzymatic activity were furtherpurified through gel filtration chromatography (Superdex 200 10/30,Pharmacia). A resultant fraction which showed an electrophoreticallysingle band (semi-purified enzyme) was then purified again by columnchromatographic focusing (Mono P 5/20 column, Pharmacia), using 0.025 Mbis-Tris-HCl (pH 6.3) as starting buffer, and polybuffer 74-HCl (pH 3.8)diluted 10 times as eluate.

The major conditions for the purification of the enzyme were as follows:

TABLE 5 Solution Total Enzyme Yield (ml) Activity (Unit) (%)Freeze-dried extent 7700 219,000 100 Ultrafilitration 1200 228,600 104Anion exchange 130 214,200 98 chromatography Gel filtration 15 657 0.3chromatography chromatographic 8 325 0.15 focusing

(2) Molecular Weight and Isoelectric Point

The molecular weight of the holoenzyme as found by gel filtration wasapproximately 360 to 380 kDa.

The result of SDS-PAGE suggested the existence of a subunit with amolecular weight of approximately 54 kDa.

The glycoside chain was removed by a method adapted from Muramatsu, etal. (Methods in Enzymology, 50, 555 (1987)) as follows: First, 1.2 times(w/w) of SDS per 1 μg enzyme was added to denature the enzyme in hotwater bath; Then, with 1 milliunit of Endoglycosidase-β-H per 1 μg ofdenatured protein added, the protein was incubated in 50 mM phosphatebuffer (pH 6.0) at 37° C. for at least 12 hours; The result proved theexistence of a subunit whose molecular weight was approximately 51 kDawith the glycoside chain removed.

The results of two-dimensional electrophoresis and hydrophobicchromatography showed that the estimated isoelectric point of the enzymewas 3.8 to 3.9.

Example D2

Enzyme which had been prepared as described in Example D1 was added to10 L of sucrose solution adjusted to a concentration of 55 wt %, pH 9.8,and a temperature of 40° C., at a rate of 20 units per 1 g of sucrose,and allowed to react at a temperature of 40° C. and pH 9.8 for 9 hours.Then, activated carbon (Taiko activated carbon S-W50) was added at arate of 1.0% of the solids content to deactivate the enzyme anddecolorize the solution at a temperature of 95° C. for 30 minutes.

The sugar composition of the solution was 55% of 1-kestose, 16% ofsucrose, 25% of monosaccharides, and 4% of nystose.

The deactivated solution was filtered to remove any impurities such asthe activated carbon. Then, the filtrate was decolorized and desaltedthrough activated carbon and ion exchange resin, then filtrated again tomake a colorless and transparent 1-kestose solution.

In order to improve the purity of 1-kestose in the 1-kestose solutionobtained above, the solution was chromatographically separated by atwo-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 95% of 1-kestose,4% of sucrose, and 1% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).

Example D3

Enzyme which had been prepared as described in Example D1 was added to10 L of sucrose solution adjusted to a concentration of 50 wt %, pH 7.0,and a temperature of 35° C., at a rate of 2 units per 1 g of sucrose,and allowed to react at a temperature of 35° C. and pH 7.0 for 60 hours.Then, activated carbon (Taiko activated carbon S-W50) was added at arate of 0.3% of the solids content to deactivate the enzyme anddecolorize the solution at a temperature of 95° C. for 30 minutes.

The sugar composition of the solution was 53% of 1-kestose, 17% ofsucrose, 25% of monosaccharides, and 5% of nystose.

The deactivated solution was filtered to remove any impurities such asthe activated carbon. Then, the filtrate was decolorized and desaltedthrough activated carbon and ion exchange resin, then filtrated again tomake a colorless and transparent 1-kestose solution.

In order to improve the purity of 1-kestose in the 1-kestose solutionobtained above, the solution was chromatographically separated by atwo-component simulated-moving bed chromatographic separator. Achromatographic fraction with a sugar composition of 90% of 1-kestose,3% of sucrose, and 7% of nystose was obtained.

Then, the chromatographic fraction was decolorized and desalted to makea colorless and transparent 1-kestose fraction (crystallizing sample).The. crystallizing sample thus prepared was treated in the sameprocedure as in Example B4 described above, to finally make similarcrystal 1-kestose.

What is claimed is:
 1. A process for producing crystal 1-kestosecomprising the steps of: (a) concentrating a highly pure 1-kestosesolution having 80% or more of 1-kestose to a Brix of at least 75,adding seed crystals, and then heating the resultant concentrate to atemperature of at least 60° C. to allow the crystals to grow, (b)concentrating the concentrate under a reduced pressure to allow thecrystals to grow, (c) heating the concentrate to redissolvemicrocrystals which have been generated in the concentrate and, if themicrocrystals fail to redissolve completely, adding water to theconcentrate to dissolve the microcrystals, and (d) repeating at leastonce steps (b) and (c) and recovering the crystal 1-kestose.
 2. Aprocess for producing crystal 1-kestose comprising the steps of: (a′)concentrating a highly pure 1-kestose solution having 80% or more of1-kestose to a Brix of at least 75, and heating the resultantconcentrate to a temperature of at least 60° C., (b′) concentrating theconcentrate under a reduced pressure to lower the temperature of theconcentrate and to initiate crystallization and allow the resultantcrystals to grow, (c′) heating the concentrate in accordance with step(c) of claim 1, and (d′) repeating at least once steps (b) and (c) ofclaim 1, and recovering the crystal 1-kestose.
 3. The process forproducing crystal 1-kestose according to claim 1 or 2, wherein theconcentrate having a Brix of 75 to 85 is concentrated under a reducedpressure.
 4. The process for producing crystal 1-kestose according toclaim 1 or 2, wherein the concentrate is concentrated in step (b) at anabsolute pressure of 40 to 200 mmHg and a concentrate temperature of 40°C. to 70° C.
 5. The process for producing crystal 1-kestose according toclaim 1 or 2, wherein the concentrate is heated to 70° C. to 95° C. instep (c) to dissolve the microcrystals.
 6. A process for producingcrystal 1-kestose comprising the steps of: (α′) concentrating a highlypure 1-kestose solution having 80% or more of 1-kestose to a Brix of atleast 80, maintaining the temperature of 50° C. to 60° C. to initiatecrystallization, then maintaining the resultant crystals at 70° C. to95° C. to grow the crystals, (β′) cooling the concentrate by 5° C. to20° C. from the temperature in step (α′), (γ′) maintaining theconcentrate at the lowered temperature in step (β′) to allow thecrystals to grow, and (δ′) repeating at least once steps (β′) and (γ′),lowering the temperature to 20° C. to 60° C., and recovering the crystal1-kestose.
 7. The process for producing crystal 1-kestose according toclaim 6, wherein the 1-kestose solution in step (α′) has 90% or more of1-kestose.
 8. The process for producing crystal 1-kestose according toany one of claims 1, 2 or 6, wherein the 1-kestose solution in step (a),(a′) or (α′) has a nystose content of 10% or less.
 9. The process forproducing crystal 1-kestose according to any one of claims 1 or 6,wherein the crystal 1-kestose is recovered at room temperature.
 10. Acontinuous process for producing crystal 1-kestose comprising performingat least twice the process according to any one of claims 1, 2 or 6 tocontinually recover crystal 1-kestose from the 1-kestose solution.
 11. Acrystal 1-kestose obtained by the process according to any one of claims1, 2 or 6, wherein the crystal 1-kestose is in the form of a columnarcrystal having a length of 0.3 to 2 mm and a purity of at least 95%. 12.A crystal 1-kestose, in the form of a columnar crystal, having a lengthof more than 1.0 mm and a purity of at least 95%.
 13. A crystal1-kestose, in the form of a columnar crystal, having a length of morethan 1.0 mm but not exceeding 1.2 mm and a purity of at least 95%. 14.The crystal 1-kestose according to claim 11, in the form of a columnarcrystal, having a length of about 0.5 mm to 2 mm.
 15. The crystal1-kestose according to claim 11, in the form of a columnar crystal,having a length of about 1.0 mm to 1.2 mm.
 16. The crystal 1-kestoseaccording to claim 12, wherein the length of the crystal 1-kestose doesnot exceed 2.0 mm.